This invention relates to a method and apparatus utilizing a continuous-gradient agar diffusion technique for determining a drug concentration parameter. More particularly, this invention relates to such a method and apparatus for determining a minimum inhibitory concentration of an antimicrobial drug required to substantially prevent the growth of a bacterium. In addition, or alternatively, the drug concentration parameter determined by the method and apparatus of this invention may be an unknown concentration of an antimicrobial drug taken, for example, from a body fluid or tissue sample or from a laboratory preparation.
Most clinical microbiology laboratories in the United States and in other countries throughout the world use the agar disk-diffusion method for determining the susceptibility of microorganisms, i.e., bacteria, to various antimicrobial drugs. In the agar disk-diffusion method, a sample of a bacterium, for example, from a body fluid of a patient is placed on the surface of an agar nutrient medium along with several small absorbant paper disks each containing a predetermined amount of a respective antimicrobial drug. The bacteria sample is then permitted to incubate for a number of hours to form a bacterium culture on the surface of the agar medium. During the incubation period, the drugs in the paper disks diffuse outwardly therefrom through the agar medium. If the bacterium being tested is susceptible to a particular antimicrobial drug, the growth of the bacterium is substantially prevented or inhibited within a circular zone surrounding the respective drug-carrying disk, the radius or diameter of the inhibition zone being a function of the degree of susceptibility of the bacterium to the particular drug.
The results of conventional methods utilizing the disk diffusion technique are invariably qualitative. The degree of susceptibility of a tested bacterium to an antimicrobial agent is characterized as "susceptible," "moderately susceptible," "intermediate" or "resistant" by a medical technician, depending on the relative size of the inhibition zone surrounding the disk carrying the antimicrobial agent. For example, if the diameter of the bacteria-growth inhibition zone for a particular drug is relatively small, the bacterium under study is characterized as "resistant." In contrast, if a bacteria-growth inhibition zone surrounding a disk is relatively large, the bacterium under study is characterized as "susceptible."
In another method for determining the susceptibility of a bacterium to an antimicrobial drug, known as the "dilution" method, samples of the bacterium under test are placed in respective nutrient solutions which contain respective concentrations of the antimicrobial drug. The drug concentrations are varied in a regular fashion. The dilution method yields a quantitative value of a minimum inhibitory concentration of the antimicrobial drug with respect to the tested bacterium. This minimum inhibitory concentration is the least concentration of the antimicrobial drug which substantially prevents or inhibits the growth of the bacterium. The minimum inhibitory concentration is determined, in accordance with the dilution method, by simply identifying the receptacle with the least drug concentration which exhibits no bacterial growth.
The dilution method is advantageous in that it provides quantitative antimicrobial susceptibility test results. However, the dilution testing method is usually substantially more expensive than the disk diffusion method, inasmuch as quantitative dilution testing requires expensive materials, expensive instrumentation and retraining of laboratory personnel. Moreover, in practice, the number of concentrations tested is often reduced in order to make room for the testing of a greater number of antimicrobial drugs on a microdilution panel (an array of solution receptacles). It has also been discovered that the quality of the results from the dilution testing method is difficult or impossible to control if the number of dilutions per drug is reduced substantially.
It is to be noted that the dilution method measures bacterial susceptibility on a discrete scale, interrupted by dilution intervals, while the disk diffusion method is capable of measuring bacterial susceptibility on a continuous scale. Thus, the disk diffusion method provides a potential for greater accuracy and reliability in the measurement of bacteria susceptibility to antimicrobial agents than is possible with the dilution method.
Correlation coefficients between zone diameters determined by the disk diffusion method and minimum inhibitory concentrations determined by the dilution method are published for most antimicrobics early in their development. The coefficients clearly demonstrate a close correlation between the disk diffusion method and a painstakingly executed dilution method performed with small steps between the dilutions.
In reports on the results of disk diffusion tests, the terms "susceptible" and "resistant" are used without reference to a specific concentration of the antimicrobial agent under consideration. The terms mislead many to believe that bacteria are inherently susceptible or resistant to all drugs. Moreover, the qualitative nature of these terms allows no distinction between varying degrees of susceptibility and makes it exceeding difficult for a clinician to know whether he is likely to achieve a concentration of the drug that will exceed the minimum inhibitory concentration in the body tissue harboring an infection.
The "breakpoints" in the discontinuous qualitative categories of conventional disk diffusion test results are determined essentially arbitrarily. Widespread disagreement over which "breakpoints" to use results in widespread discrepancies in results reported to physicians.
Each antimicrobial chemical has a usual concentration in each of the major body fluids, based on a given usual drug dosage, i.e., in blood, the bile, the urine and the cerebral spinal fluid. The ratio between the usual concentration at the lowest usual dosage of an antimicrobial drug in a body fluid to the minimum inhibitory concentration of that drug for a particular bacterium provides an especially useful quantitative indication of the expected effectiveness of that drug in combating the presence of the bacterium in that body fluid. This ratio is called an "inhibitory quotient." The higher the inhibitory quotient, the more likely it is that the respective drug will be successful in eradicating the existence of the invading microorganism in the respective body fluid or in a body tissue capable of sustaining a drug concentration predictably related to the maximum concentration in the body fluid. Thus, inhibitory quotients enable the physician to determine the probability that the concentration of a particular potentially administerable antimicrobial will exceed the minimum inhibitory concentration at the site of the infection. The calculation of inhibitory quotients facilitates the comparison of the relative activity of various drugs in different body tissues. Generally, higher dosages result in higher inhibitory quotients which indicate higher tissue concentrations.
An object of the present invention is to provide a method and an associated apparatus which use the agar diffusion technique to determine a quantitative drug concentration parameter.
Another, more particular, object of the present invention is to provide such a method and such an associated apparatus for quantitatively gauging the susceptibility of a microorganism such as a bacterium to an antimicrobial agent.
Another particular object of the present invention is to provide such a method and such an apparatus for determining unknown concentrations of particular antimicrobial drugs.
Another object of the present invention is to provide such a method and such an apparatus which are inexpensive and easy to use.
A more particular object of the present invention is to provide such a method and such an apparatus for making quantitative determinations of minimum inhibitory concentrations of one or more drugs with respect to a microorganism and for making further quantitative determinations of inhibitory quotients.